Wu et al. Methods 2014, 10:19 http://www.plantmethods.com/content/10/1/19 PLANT METHODS

METHODOLOGY Open Access AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings Hung-Yi Wu1,2, Kun-Hsiang Liu3,4, Yi-Chieh Wang1, Jing-Fen Wu1, Wan-Ling Chiu3,4,5, Chao-Ying Chen2, Shu-Hsing Wu1, Jen Sheen3,4 and Erh-Min Lai1,2,3,4*

Abstract Background: Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. Results: We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using β-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter- gene regulation as well as protein subcellular localization and protein–protein interactions in physiological contexts. Conclusions: AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein–protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer. Keywords: Agrobacterium, Arabidopsis, Transient transformation, Gene expression, Innate immunity, Gain-of-function

Background transient gene expression via Agrobacterium-mediated Agrobacterium-mediated DNA transfer is currently the DNA transfer in different plant tissues offers a simple and most facile and versatile method to deliver gene constructs fast method to analyze transgene functions, which is into the nucleus for gene function analysis in diverse plant amenable for high-throughput screens. The transient ex- species [1-3]. Although stable integration of physiologically pression assay is also ideal for systematic dissection of the active and regulated transgenes is the ultimate goal, exquisite and complex processes of Agrobacterium–plant interactions and DNA transfer events [4-7]. * Correspondence: [email protected] Agrobacterium tumefaciens is a soil phytopathogen that 1Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, naturally infects plant wound sites and causes crown gall Taiwan disease via delivery of transferred (T)-DNA from bacterial 2Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan cells into host plant cells through a bacterial type IV Full list of author information is available at the end of the article

© 2014 Wu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wu et al. Plant Methods 2014, 10:19 Page 2 of 16 http://www.plantmethods.com/content/10/1/19

secretion system (T4SS) [8]. Although Agrobacterium is Pattern-triggered immunity (PTI) induced by a microbe- considered a wound-associated pathogen, it can transfer or pathogen-associated molecular pattern (MAMP or DNA into diverse host cells or tissues under unwounded PAMP) is the first line of active defense in both and conditions [9-13]. Interestingly, most of the Arabidopsis animals against pathogens [28-30]. Previous studies have mutants that are resistant to Agrobacterium transformation suggested that Agrobacterium-mediated transformation effi- identified by root explant assays remain highly transform- ciency may be compromised when plants recognize Agro- able by floral dip transformation [14]. The mechanisms and bacterium MAMPs by corresponding pattern-recognition plant factors involved in Agrobacterium-mediated trans- receptors (PRRs) to trigger PTI and block Agrobacterium formation may differ between wounded and unwounded infection [22,24]. The elongation factor Tu (EF-Tu) recep- cells or different tissues. However, the mechanisms under- tor mutant efr-1, which cannot sense EF-Tu MAMP, lying Agrobacterium infection in unwounded cells/tissues showed increased Agrobacterium-mediated transient ex- have not been explored. pression efficiency, as did transgenic Arabidopsis express- In plant biology research, Arabidopsis mesophyll- ing a potent bacterial effector AvrPto to suppress PTI transfection [15,16] and Agrobacterium-medi- signaling with agroinfiltration of 4- to 5-week-old leaves ated leaf infiltration in Nicotiana benthamiana [17] are [22,24]. However, whether these immune-compromised the well-established and commonly used platforms for Arabidopsis plants are amenable to increase Agrobacterium- transient gene expression analysis. The Arabidopsis mediated transient expression efficiency in young seed- mesophyll-protoplast transient expression system allows lings has not been tested. Defining the condition for for versatile and high-throughput analyses of diverse reliable and highly efficient transformation in healthy gene functions and signal transduction pathways; ad- Col-0 seedlings will be extremely valuable but has never vanced skills with training and practice are essential for been achieved. successful use of this powerful tool for gene function In this study, we systematically investigated various bio- studies [16,18,19]. Agrobacterium-mediated transient ex- logical factors and growth variances to define a combin- pression methods by leaf infiltration have been devel- ation of key factors that contribute to the unprecedentedly oped for a wide range of plants including Nicotiana, high transient transformation and reporter gene expression lettuce, tomato, and Arabidopsis [20-23]. However, the efficiency in Arabidopsis seedlings. As a result of these use of 4- to 5-week-old adult plants with manual infiltra- investigations, we developed an optimized AGROBEST tion has limited application in high-throughput analyses. (Agrobacterium-mediated enhanced seedling transform- Furthermore, although Arabidopsis is the most-studied ation) method that enabled high transient transform- model plant with superbly annotated genome sequences ation and expression efficiency in both efr-1 mutant and and powerful genetic and genomic resources mostly Col-0 Arabidopsis seedlings. Importantly, we demon- available for the Columbia (Col) accession, achieving strated the versatile applicability of AGROBEST in gain- highly efficient and consistent transient expression in of-function studies for the MYB75 transcription factor Col by adult leaf infiltration is challenging [22,24]. in specific target-gene activation and for GIGANTEA (GI) The use of young seedlings for Agrobacterium-mediated reporter gene expression regulated by the Arabidopsis cir- transient expression assays will greatly simplify and cadian clock. The AGROBEST method is a fast, simple, amplify the power of the method. Indeed, Agrobacterium- reliable, and versatile tool for systematic gene function mediated transient expression in Arabidopsis seedlings analysis and a new tool for dissecting the Agrobacterium- has been recently developed for fast and robust analysis mediated DNA transfer processes. of protein subcellular localization and protein–protein interactions [25-27]. The system’s requirement for high- Results density Agrobacterium cells and vacuum infiltration [27] Cotyledons of young Arabidopsis EF-TU receptor mutant or chemical treatment (e.g., the addition of surfactant is highly susceptible to Agrobacterium-mediated transient Silwet L-77) [26] to achieve high cellular transformation transformation efficiency could induce innate immunity and stress re- Environmental and biological factors such as growth sponses in plants, which globally alters cellular, physio- conditions, host plants, and Agrobacterium strains can logical, and signaling processes and severely retards affect the transformation efficiency. We first evaluated growth [28,29]. Thus, developing a system that circum- the transient expression efficiency of selected Arabidopsis vents a plant defense barrier may be a key to enhance ecotypes and mutants defective in pattern-recognition transient expression efficiency in Arabidopsis seedlings. receptors (PRRs) with a disarmed A. tumefaciens strain Furthermore, such a fast, robust, and highly efficient C58C1(pTiB6S3ΔT-DNA) [31] containing a pCH32 helper transient expression system could support gain-of- [32] and abbreviated as C58C1(pTiB6S3ΔT)H.The function studies of diverse genes and signaling pathways T-DNA vector pBISN1 harboring the gusA-intron [12] was in planta. transformed into C58C1(pTiB6S3ΔT)H to infect 4-d-old Wu et al. Plant Methods 2014, 10:19 Page 3 of 16 http://www.plantmethods.com/content/10/1/19

seedlings, and β-glucuronidase (GUS) activity was deter- mined to monitor transient expression efficiency at 3 days post-infection (dpi). We consistently observed 100% of ana- lyzed EF-Tu receptor mutant efr-1 seedlings were success- fully transformed, with strong and homogenous GUS staining in cotyledons, with 4-fold higher GUS activity in efr-1 than wild-type Col-0 seedlings (Figure 1A and B, Additional file 1: Table S1). The flagellin receptor mutant, fls2, and the Ws ecotype that is highly susceptible to Agrobacterium transformation in the root explant [14] and anaturalfls2 variant [33] showed similar transient GUS ex- pression efficiency as the Col-0, so the fls2 mutant contrib- utes little to enhancing Agrobacterium-mediated transient transformation (Figure 1A and B). Our seedling transient expression results confirm and further support that EFR but not FLS2 is an important factor limiting Agrobacterium- mediated transient expression efficiency previously observed by agroinfiltration of Arabidopsis adult leaves [24,34,35].

Buffered medium at pH 5.5 with AB salts is critical for high transient expression efficiency To exploit this transient expression system for higher effi- Figure 1 Transient transformation assays in different ciency, we tested several factors including pre-induction Arabidopsis ecotype/genotypes. Four-day-old Arabidopsis seedlings were infected with Agrobacterium strain C58C1(pTiB6S3ΔT)H carrying and co-cultivation conditions. Pre-induction with acetosyr- pBISN1, which was pre-incubated in AB-MES (pH5.5) supplemented ingone (AS) in AB-MES medium (ABM50 and ABM200 with 200 μM acetosyringone (AS) to induce vir gene expression. methods) and continuous addition of AS to stimulate vir Seedlings were co-cultivated with pre-induced A. tumefaciens cells gene expression during the infection process are required with final OD600 = 0.02 in the MS medium (1/2 MS, 0.5% sucrose (w/v), μ for efficient transient GUS expression. Because AB-MES pH 5.5) containing 50 M AS and determined for transient GUS expression levels by overnight GUS staining (A) and quantitative medium (pH 5.5) is the optimized medium for vir gene GUS activity (B) at 3 dpi. The GUS activity obtained from Col-0 induction [36,37], we tested whether mixing AB-MES seedlings was set to 100% and that of Ws, efr-1,andfls2 is relative (pH 5.5) with an equal volume of commonly used plant to that of Col-0. Data are mean ± SD GUS activity from two biological culture MS medium (1/2 MS, 0.5% sucrose (w/v), replicates. Similar results were obtained from at least two independent pH 5.5), named ABM-MS (1/2 AB-MES, 1/4 MS, 0.25% experiments. Values significantly different from that obtained with Col-0 are denoted (*P = 0.058 by Student’s t test). sucrose (w/v), pH 5.5) in the presence of AS could pro- duce high transient expression efficiency. Strikingly, GUS activity was strongly expressed in all seedlings and sodium phosphate at pH 5.5 showed comparable and was 20-fold higher with co-cultivation in ABM-MS than strong GUS activity as that with ABM-MS (Figure 2C). in MS medium alone (Figure 2A, Additional file 1: Table S1). However, omitting AB salts resulted in ~50% reduction To avoid over-staining, the reaction time for histological in GUS activity, and no GUS activity was detected with GUS staining shown in Figure 2 was limited to 6 hr MS medium buffered with sodium phosphate at pH 7.0 instead of overnight for the result in Figure 1A. in the presence or absence of AB salts. Thus, buffered Key components in AB-MES are AB salt (17.2 mM pH at 5.5 and the presence of AB salts in MS co- K2HPO4, 8.3 mM NaH2PO4, 18.7 mM NH4Cl, 2 mM cultivation medium are the two key factors for this high KCl), minerals (1.25 mM MgSO4, 100 μM CaCl2,10μM transient expression efficiency. We named this opti- FeSO4), glucose (2% w/v), and buffering with MES mized infection method AGROBEST (Agrobacterium- (50 mM) to pH 5.5. We thus tested whether one of these mediated enhanced seedling transformation). components is responsible for the increased transient expression efficiency. The addition of AB salts with MES Disarmed Agrobacterium strain C58C1(pTiB6S3ΔT)H buffered at pH 5.5 in MS medium was sufficient to re- enables highly efficient AGROBEST-mediated transient sult in comparable levels of GUS expression as with expression in Col-0 seedlings ABM-MS (Figure 2B). Therefore, AB salts alone, pH 5.5 Next, we tested whether the AGROBEST method optimized buffered by MES, or both, are critical for the increased with efr-1 seedlings could also improve Agrobacterium- transient expression efficiency. Strikingly, all MS media mediated transient transformation in wild-type Col-0 with the addition of AB salts buffered with MES or seedlings. Because the use of C58C1(pTiB6S3ΔT)H as Wu et al. Plant Methods 2014, 10:19 Page 4 of 16 http://www.plantmethods.com/content/10/1/19

Figure 2 Optimization of Agrobacterium pre-culture and infection media for efficient transient expression efficiency. Four-day-old Arabidopsis efr-1 seedlings infected with Agrobacterium C58C1(pTiB6S3ΔT)H carrying pBISN1 were grown in various pre-culture and co-cultivation media to test their effects on transient GUS expression efficiency measured by GUS staining and quantitative GUS activity. (A) Various pre-culture and infection media in the absence or presence of vir gene inducer AS at the indicated concentration. (B) Effect of factors in AB-MES medium on increased transient expression efficiency. (C) Effect of AB salts, pH and buffering systems on transient GUS expression efficiency. Data for relative quantitative GUS activity are mean ± SD of 3 independent experiments. Values significantly different from that infected by ABM50 (A) or condition 1 (B and C) are denoted (*P <0.05,**P < 0.01, ***P < 0.005 by Student’s t test). compared with other disarmed or virulent A. tumefaciens expression efficiency by AGROBEST than ABM50 (Figure 3A strains produced higher transient expression levels and B). Moreover, Col-0 seedlings infected by AGROBEST with leaf agroinfiltration of various plants [23], we also showed higher transient expression than efr-1 seedling tested whether C58C1(pTiB6S3ΔT)H isamoresuperior infected by ABM50 (Figure 3A and B). No GUS stains strain in our system. We compared C58C1(pTiB6S3ΔT)H could be detected in control seedlings without infection with the wild-type virulent strain C58 or C58-derived (MOCK) or infected with ΔvirB2, a strain lacking the key disarmed strain GV3101(pMP90) [38] for their transient component of the type IV secretion system (T4SS) essen- expression efficiency in efr-1 and Col-0 seedlings using both tial for T-DNA/effector translocation [8,39]. Therefore, sub-optimal ABM50 and optimized AGROBEST methods. the GUS activity detected was indeed from T-DNA gene Remarkably, Col-0 seedlings infected by all transfer- expression inside the plant cells. Strikingly, 5- to 15-fold competent strains achieved significantly higher transient higher GUS activity was observed in efr-1 or Col-0 Wu et al. Plant Methods 2014, 10:19 Page 5 of 16 http://www.plantmethods.com/content/10/1/19

Figure 3 Transient transformation of the Arabidopsis seedlings by various Agrobacterium strains. Four-day-old Arabidopsis Col-0 and efr-1 seedlings infected with different Agrobacterium stains carrying pBISN1 by ABM50 or ABM-MS (named as AGROBEST) were compared by GUS staining (A), quantitative GUS activity (B), and root length (C) at 3 days post-inoculation (dpi). Data for quantitative GUS activity are mean ± SD of at least 4 biological replicates from 2 independent experiments. Values significantly different from that infected with wild-type C58 are denoted (*P <0.05, **P <0.01byStudent’s t test). Data for root length measurement are mean ± SEM of 4-6 biological replicates from 2–4 independent experiments. Statistics was analyzed by ANOVA and means annotated with the same letter (a-c) are not significantly different; those with different letters are significantly different (P < 0.05). Seedlings grown in the same co-cultivation medium without Agrobacterium infection are indicated (MOCK). seedlings infected with C58C1(pTiB6S3ΔT)H than with efficiency in both Col-0 and efr-1 seedlings. Remarkably, C58 or GV3101(pMP90) (Figure 3A and B). The root all Col-0 seedlings infected by the AGROBEST showed length was significantly shorter for Arabidopsis seedlings ~10-fold increased transient expression efficiency than infected with C58, ΔvirB2, or GV3101(pMP90) at 3 dpi in with two existing methods, the FAST method [26] and all combinations or with C58C1(pTiB6S3ΔT)H by the the method by Marion et al. [27] with either GUS AGROBEST method as compared with uninfected seed- (Figure 4A) or luciferase (LUC2) (Figure 4B) used as re- lings (MOCK) (Figure 3C). Notably, seedlings infected porters. Interestingly, both AGROBEST and the Marion with C58C1(pTiB6S3ΔT)H showed no or little inhibition et al. method achieved significantly higher transient ex- of root elongation in efr-1 seedlings with the AMB50 pression activity in efr-1 than in Col-0, efr-1 seedlings method, which achieves fair although not the highest tran- remained poorly transformed by the FAST method sient expression efficiency. (Figure 4A and B). As a result, AGROBEST conferred at least 40-fold and 3-fold higher transient expression effi- AGROBEST achieves higher transient expression efficiency ciency in efr-1 seedlings than with FAST and the Marion than existing methods in both efr-1 and Col-0 seedlings et al. methods, respectively (Figure 4A and B). However, We also compared AGROBEST with previously devel- we detected no increased transient expression activity in oped methods [26,27] for their transient expression infected seedlings of the dexamethasone (DEX)-induced Wu et al. Plant Methods 2014, 10:19 Page 6 of 16 http://www.plantmethods.com/content/10/1/19

Figure 4 AGROBEST enables high transient expression levels in Col-0. Four-day-old Arabidopsis seedlings were infected with Agrobacterium strain C58C1(pTiB6S3ΔT)H carrying pBISN1 (A and C) or 35S::LUC2 (B), and transient expression activity was determined at 3 dpi. (A) Transient GUS expression efficiency of Col-0 and efr-1 seedlings by AGROBEST, FAST and Marion et al. methods. Data for quantitative GUS activity are mean ± SD of 3 biological replicates. Values significantly different from those obtained with Col-0 by AGROBEST are denoted (*P < 0.05 by Student’s t test). (B) Transient luciferase expression efficiency of Col-0 and efr-1 seedlings by AGROBEST, FAST and Marion et al. methods. Seedlings infected by C58ΔvirB2 carrying 35S::LUC2 were used as a background control and those without Agrobacterium infection are indicated as MOCK. Luciferase activity of Col-0 obtained by AGROBEST was set to 100% and that of others is relative to activity of Col-0 by AGROBEST. Data are mean ± SD of 3 biological replicates. Values significantly different from those obtained with Col-0 by AGROBEST are denoted (**P < 0.01, by Student’s t test). (C) Transient GUS expression efficiency of Col-0, AvrPto transgenic line, and efr-1 by AGROBEST. For dexamethasone (DEX) treatment, 3-d-old seedlings were treated with 10 μM DEX for 1 day and the following 3 days infected by the AGROBEST method. Quantitative GUS activity from DEX-induced Col-0 seedlings by AGROBEST was set to 100% and that of others is relative to activity of DEX-induced Col-0 seedlings with AGROBEST. Data are mean ± SD GUS activity from 4 repeats (2 biological repeats from each of 2 independent experiments). Values significantly different from that obtained with Col-0 are denoted (**P <0.01byStudent’s t test). Seedlings grown in the same co-cultivation medium without Agrobacterium infection are indicated (MOCK).

AvrPto transgenic line than in Col-0 seedlings with the Impact of seedling age and infection time on transient AGROBEST method (Figure 4C), despite a significantly expression efficiency of AGROBEST in efr-1 seedlings higher transient expression efficiency than Col-0 de- Because the highest transient expression efficiency in tected in adult leaves by agroinfiltration [22]. The efr-1 seedlings can be achieved by infection with C58C1 AvrPto transgenic line germinated at the same rate and (pTiB6S3ΔT)H by AGROBEST, we chose this combin- grew to a similar size as Col-0 and efr-1, but growth was ation to test the versatility and applicability of AGROB- arrested with the addition of DEX at 3 days old. This EST. For example, dissecting the minimal infection time finding is consistent with previous studies showing that (from 1–5 days) and range of seedling age (from 3- to 6- overexpression of AvrPto can also interfere with growth d-old) applicable for efficient transient expression is of hormone signals and trigger cell death by interrupting interest. We tested different ages of Arabidopsis efr-1 seed- the diverse functions of BAK1 and BKK1 in multiple re- lings infected at different dpi and noted that GUS signals ceptor complexes, not restricted to PRRs [40]. were barely detectable at 1 dpi but gradually reached a Wu et al. Plant Methods 2014, 10:19 Page 7 of 16 http://www.plantmethods.com/content/10/1/19

plateau at 3 or 4 dpi (Figure 5A). When 5- or 6-d-old seedlings were infected, we observed transient GUS ex- pression in true leaves at 3 or 4 dpi. Although strong GUS staining could still be detected in seedlings at 4 or 5 dpi, these seedlings often showed bleached lesions in cotyledons (Figure 5B), which explained the lack of GUS expression in part of the cotyledons at 4 or 5 dpi (Figure 5A). At 3 dpi, the bleached lesions were more visible when the transformation was performed with 5- or 6-d-old seedlings than with 3- or 4-d-old seed- lings. Thus, the use of younger seedlings for AGROB- EST may be more desirable for maintaining plants in healthy and physiological conditions. To test the minimal infection time for GUS detection and to avoid plant damage due to prolonged Agrobacterium infection, the co-cultivation medium was replaced with fresh medium containing antibiotics (100 μM Timentin) at 1 or 2 dpi to inhibit bacterial growth. In 4-d-old seedlings, we detected low levels of GUS signals with an additional 2 or 3 days of cultivation after Timentin treatment at 1 dpi (Figure 6). Importantly, with Timentin treatment at 2 dpi, seedlings with 1 to 3 days of additional cultivation remained healthy (without bleached lesions) and showed strong GUS signals in cotyledons. Because Agrobacterium cells were mostly killed when true leaves emerged from infected seed- lings,thenewlygrowntrueleaves were not efficiently trans- formed. Therefore, the use of 4-d-old seedlings infected for 2 days followed by an additional 1 to 3 days of cultivation with Timentin is the optimal condition to transiently ex- press genes for functional studies.

Widespread transient transformation events in different organs and cell types Figure 5 Impact of seedling age and infection time on The high transient expression efficiency with AGROB- transient expression. (A and B) Different ages of Arabidopsis efr-1 EST was mostly evident with strong and homogeneous seedlings were infected with C58C1(pTiB6S3ΔT)H carrying pBISN1 by GUS signals detected in cotyledons of 100% infected the AGROBEST method and analyzed for GUS activity (A) and Col-0 or efr-1 seedlings (Figures 3A, 4A and 7A, Add- morphologic features (B) at different dpi. itional file 1: Table S1). When 7-d-old seedlings were used for infection, strong GUS signals were also detected system allows for high transient gene expression and, in true leaves, as shown in efr-1 seedlings (Figure 7B). potentially, functional analysis in diverse tissues and cell However, in roots, GUS signals could be detected in ~70% types in Arabidopsis seedlings. of Col-0 or efr-1 seedlings infected by AGROBEST (Additional file 1: Table S1) and mostly appeared in lateral Studies of protein subcellular localization and protein– root initiation sites or in the elongation zone (Figure 7C protein interactions and D). In addition to analyzing the GUS reporter, we de- Because Arabidopsis plants are less amenable for transient termined the expression of fluorescent proteins as reporters expression analysis, both fluorescent protein localization at cellular and subcellular levels using efr-1 seedlings. With and bimolecular fluorescence complementation (BiFC) expression of the Venus-intron or NLS-RFP driven by the studies are often conducted in via transfection CaMV 35S promoter, fluorescent protein signals were or in N. benthamiana leaves via agroinfiltration because widely detected in cotyledon cells (Figure 7E and F), mainly of the high transient expression efficiency [16,17]. Here, in epidermal pavement cells but also in guard cells and we co-infected two A. tumefaciens strains carrying a bin- mesophyll cells (Figure 7G-I). For roots, epidermal cells ary vector for 35S::Venus-intron or 35S::NLS-RFP in efr-1 consistently showed fluorescent protein signals (Figure 7J). seedlings and detected both cytoplasmic and nuclear Therefore, the AGROBEST seedling transformation fluorescence signals for Venus and nuclear localization of Wu et al. Plant Methods 2014, 10:19 Page 8 of 16 http://www.plantmethods.com/content/10/1/19

monitor real-time bioluminescence for 5 days. In contrast to constant low levels of bioluminescence from seedlings infected with a vector control, Arabidopsis seedlings infected with Agrobacterium delivering GI::LUC2 showed clear circadian oscillation at slightly length- ened period for at least 5 days (Figure 8). The observed transiently expressed GI circadian cycle is indistin- guishable from the stable GI expression in GI::LUC2 transgenic Arabidopsis plant (TP) [43], although with lower amplitude. The comparable circadian oscillation between the stable and transient expression of the GI:: LUC2 indicated that the slightly longer period we observed was unlikely a result of the Agrobacterium infection. This result indicated the applicability of AGROBEST for transient expression of circadian rhythm reporter in Arabidopsis seedlings without detectable inter- ference by Agrobacterium infection.

AGROBEST for functional assays of transcription factor MYB75 Figure 6 Impact of Timentin treatment on transient GUS Next, we tested AGROBEST for gain-of-function studies. expression efficiency. Four-day-old Arabidopsis efr-1 seedlings were For a proof of concept, we transiently expressed a tran- Δ H infected with Agrobacterium C58C1(pTiB6S3 T) carrying pBISN1 by scription factor MYB75 because of its well-established the AGROBEST method at 1 or 2 dpi before Timentin treatment. GUS staining was performed at 0 to 3 days after Timentin treatment. function in anthocyanin accumulation by upregulating a key gene encoding chalcone synthase (CHS) in the anthocyanin synthesis pathway [44]. Four-day-old efr-1 Arabidopsis seedlings were infected for 3 days after NLS-RFP in separate or the same cells (Figure 7K). Timentin treatment for an additional 3 days to determine Our assay is also feasible for BiFC studies, which is the effect on MYB75 transient expression. MYB75 mRNA supported by the interaction of two known interacting level in infected seedlings was 60-, 400-, and 200-fold proteins, F-box protein TIR1 (transport inhibitor higher when the expression was driven by single (1X35S) response 1) and ASK1 (Arabidopsis Skp1-like protein) and double (2X35S) CaMV 35S promoter and super pro- [41], in the nucleus (Figure 7L). Thus, AGROBEST moter, respectively, than in seedlings expressing control is an ideal system for subcellular localization and vectors (Figure 9A). CHS mRNA level was increased 4- protein-protein interaction studies. and 3-fold in 2X35S::MYB75 and super::MYB75 seedlings, respectively (Figure 9B). However, CHS expression was AGROBEST for the expression analysis of a circadian clock not increased in 1X35S::MYB75 seedlings despite its reporter gene 60-fold higher MYB75 expression, which suggests a Encouraged by the high transient expression efficiency threshold expression level or the requirement of other with AGROBEST, we next tested its applicability in gene MYB75-modulated co-activators for CHS activation. function/regulation study in physiological contexts. Most Importantly, consistent with increased CHS expression, Arabidopsis genes express rhythmically under various high level of anthocyanin (purple coloration) was readily thermocycles, photocycles, or circadian clock conditions detectable in cotyledons of 2X35S::MYB75 and super:: [42]. Reporter genes driven by promoters of the circadian MYB75 seedlings but not in seedlings infected with a genes are commonly used to monitor the regulation of cir- vector control, 1X35S::MYB75,orsuper::gusA-intron cadian genes. To test whether circadian rhythm could be (Figure 9C). No increase of anthocyanin accumulation from monitored in transiently transformed seedlings, a circadian super::gusA-intron seedlings indicated that the specificity of reporter (GI::LUC2) constructed by fusing the promoter of the observed anthocyanin phenotype was due to the transi- the circadian gene GIGANTEA (GI) with the luciferase ent expression of MYB75 rather than a secondary effect gene (LUC2) [43] was used. Four-day-old Arabidopsis efr-1 from the infection or the overexpression of any foreign pro- seedlings were infected with Agrobacterium delivering GI:: tein. Importantly, AGROBEST also enables the transient LUC2 for 3 days under 16-h/8-h light/dark cycles and then expression of MYB75 to monitor its downstream CHS transferred to MS medium in the presence of 100 μM expression and anthocyanin accumulation in Col-0 seed- Timentin and 0.5 mM luciferin under continuous light to lings. We show that transient expression of MYB75 driven Wu et al. Plant Methods 2014, 10:19 Page 9 of 16 http://www.plantmethods.com/content/10/1/19

Figure 7 Transient transformation events in different organs and cell types. (A-D) Four-day-old (A and C-D) or 7-d-old (B) Arabidopsis efr-1 seedlings were infected with C58C1(pTiB6S3ΔT)H carrying pBISN1 by the AGROBEST method and analyzed for GUS staining. GUS staining was detected in true leaves (B, indicated by asterisk), cotyledons (A and B), main roots near lateral initiation site (C), and elongation zone (D). (E-L) Confocal microscopy of 4-day-old Arabidopsis efr-1 seedlings infected with C58C1(pTiB6S3ΔT)H carrying various vectors for transient expression of indicated fluorescent proteins by the ABM200 method. Fluorescence signals for 35S::Venus-intron or 35S::NLS-RFP were detected in cotyledons (E and F). Venus-intron signals were detected in different types of cells, including epidermal cells (G), guard cells (H), mesophyll cells (I) of cotyledon, and root epidermal cells (J). (K) Subcellular localization of Venus-intron and NLS-RFP by co-infection of 2 Agrobacterium strains expressing 35S::Venus-intron or 35S::NLS-RFP. (L) Protein–protein interaction by BiFC of nYFP-ASK1 and TIR1-cYFP. Images show fluorescence alone (K) and/or merged with bright field (E, F, J and L) or chloroplast fluorescence (G-I). Scale bars are 2 mm (A and B), 0.5 mm (C and D), 100 μm (E, F and J),50μm (L) and 20 μm (G-I and K). BiFC, bimolecular fluorescence complementation.

by the 2X35S promoter results in significantly higher of Discussion MYB75 mRNA levels than vector control in Col-0 AGROBEST enables high transient transformation and seedlings (Figure 10A). Remarkably, CHS mRNA levels expression efficiency in intact Arabidopsis young seedlings were also upregulated in 2X35S::MYB75 seedlings In this study, we developed a simple, fast, reliable, and (Figure 10B), in which a moderate increase in antho- robust transient expression system named AGROBEST cyanin accumulation was also detected (Figure 10C). and uncovered the key factors enabling 100% of infected This result strongly suggested the broad application of seedlings with high transgene expression efficiency in AGROBEST for gain-of-function studies not limited to Arabidopsis seedlings. Remarkably, AGROBEST appears the immune-compromised mutant. to achieve the highest transient expression efficiency in Wu et al. Plant Methods 2014, 10:19 Page 10 of 16 http://www.plantmethods.com/content/10/1/19

the EF-Tu receptor efr-1 mutant as compared to the wild-type Col-0, flagellin receptor mutant fls2, and DEX- inducible AvrPto transgenic line. This result is consistent with a previous finding in agroinfiltrated Arabidopsis adult leaves showing increased transient GUS expression efficiency in efr-1 [24]. Because of no detectable pheno- type impairing the growth and development in the efr mutant [24], the use of the efr mutant has an advantage over DEX-inducible AvrPto in seedling stages. Thus, more selected elimination of specific PRRs such as EFR with minimal effects on hormonal signaling, cell death and seedling growth may be a preferred system for Agro- bacterium-mediated high transient expression efficiency. Interestingly, N. benthamiana leaves, which are com- Figure 8 Monitoring Arabidopsis circadian rhythm by transient expression of GIGANTEA::luciferase (GI::LUC2). Four-day-old Arabidopsis monly used for Agrobacerium-mediated transient trans- efr-1 seedlings were infected with Agrobacterium C58C1(pTiB6S3ΔT)H formation, also lack the EFR receptor [45]. carrying a vector (pCAMBIA1390) or p1390-GI-LUC2 by the AGROBEST Unexpectedly, we discovered that AGROBEST also en- method for 3 days in a 16-h/8-h light/dark cycle (75 μmol m-2 s-1), then ables high transient expression efficiency in wild-type Col-0 μ transferred to 1/2 MS liquid medium in the presence of 100 M seedlings. The significantly higher transient expression ac- Timentin and 0.5 mM luciferin and grown under continuous light at 40 μmol m-2 s-1 for up to 5 days. The GI::LUC2 transgenic Arabidopsis tivity by AGROBEST than the FAST and Marion et al. plant (TP) cultured in identical conditions without Agrobacterium methods likely accounts for the success of our gain-of- infection was a positive control. Real-time bioluminescence signals were function experiments, which have not been shown previ- photographed and the luciferase intensity is shown as mean ± SEM ously [26,27]. Of note, efr-1 seedlings remained poorly from 12 seedlings expressing GI::LUC2. Similar results were obtained from transformed by FAST method as compared with the sig- at least 3 independent experiments. The white and gray regions indicate subjective light and dark periods, respectively. nificantly increased transient expression in efr-1 by AGROBEST or the Marion et al. method. The reason

Figure 9 Transient expression of MYB75 increases anthocyanin accumulation. Four-day-old Arabidopsis efr-1 seedlings were infected with Agrobacterium C58C1(pTiB6S3ΔT)H carrying a vector (pCAMBIA1390), 35S::MYB75, 2X35S::MYB75, super::MYB75, or super::gusA-intron by the AGROBEST method. At 3 dpi, co-cultivation medium was replaced with MS medium containing 100 μM Timemtin for additional incubation for 3 days. qRT-PCR of relative expression of MYB75 (A) and CHS (B) with representative data shown with mean ± SD from 3 technical repeats. Similar results were obtained from three independent experiments. Zeiss inverted microscopy of anthocyanin accumulation in seedlings (upper panels) and cotyledons (lower panels) and quantification (C). Data for anthocyanin content are mean ± SD from 4 repeats (2 biological repeats from each of 2 independent experiments, 20–30 seedlings for each biological repeat), Values significantly different from that obtained with vector are denoted (**P < 0.01, by Student’s t test). Wu et al. Plant Methods 2014, 10:19 Page 11 of 16 http://www.plantmethods.com/content/10/1/19

Figure 10 Transient expression of MYB75 increases anthocyanin accumulation in Col-0 seedlings. Four-day-old Arabidopsis Col-0 seedlings were infected with Agrobacterium C58C1(pTiB6S3ΔT)H carrying a vector (pCAMBIA1390), 2X35S::MYB75, or super::gusA-intron by the AGROBEST method. At 3 dpi, co-cultivation medium was replaced with MS medium containing 100 μM timemtin for additional incubation for 3 days. qRT-PCR of relative expression of MYB75 (A) and CHS (B) with representative data shown with mean ± SD from 3 technical repeats. Similar results were obtained from three independent experiments. Zeiss inverted microscopy of anthocyanin accumulation in seedlings (upper panels) and cotyledons (lower panels) and quantification (C). Data for anthocyanin content are mean ± SD from 3 independent experiments (20–30 seedlings for each biological repeat, 3 biological repeats for each independent experiment). Values significantly different from those obtained with vector are denoted (**P < 0.01, by Student’s t test). underlying this discrepancy is unknown, but the yellowish Widespread and differential transient transformation and retarded-growth seedlings after co-cultivation with events in different organs and cell types Agrobacterium in the dark for 2 days by the FAST method AGROBEST has a breakthrough performance by enab- may contribute to the observed phenotype. Our AGROB- ling high and homogeneous transient GUS expression EST method, applying a lower density of Agrobacterium efficiency in shoots of 100% infected Col-0 or efr-1 seed- cells (OD600 0.02 as opposed to OD600 0.5 for the FAST lings. The successful transient expression in roots, al- method and OD600 2 for the Marion et al. method) for co- though with less efficient transformation events (~70% cultivation with seedlings without any mechanical treat- of seedlings with GUS staining in roots), is also remark- ment (e.g., vacuum infiltration) or chemical treatment able and not previously detected [26,27]. Interestingly, (e.g., the addition of surfactant Silwet L-77) offers advan- preferential transformation events occurring at the initi- tages to maintain infected seedlings with normal growth ation sites of lateral roots or the root elongation zone of and a physiological state without injury. The success of infected intact seedlings were also previously detected in transiently expressing the circadian rhythm reporter in wounded Arabidopsis roots [46]. High transformation Arabidopsis seedlings may open a new platform to rapidly of Arabidopsis roots may require further loosening or test the circadian behaviors of Arabidopsis mutants, opening of cell walls or wounding, which was not bypassing the process of introducing a circadian reporter included in our infection conditions. Because we ob- gene into the mutants by crossing. Most remarkably, served similar transient expression levels and trans- AGROBEST allows for high transient expression of the formation efficiency in roots of Col-0 and efr-1 MYB75 transcription factor and subsequently upregu- seedlings (Additional file 1: Table S1), EFR may play no lates the expression of its downstream gene CHS in or little role in seedling root transformation efficiency both efr-1 and Col-0 seedlings. This result suggested the under our infection conditions. Consistently, EFR is broad application of AGROBEST to study transcription expressed at low levels in Col-0 seedling roots [47], factor action. which were not responsive to the EF-Tu peptide elf26, Wu et al. Plant Methods 2014, 10:19 Page 12 of 16 http://www.plantmethods.com/content/10/1/19

as evidenced by limited induction of immune marker genes infection than in uninfected seedlings (MOCK), despite and callose deposition in the roots of Col-0 seedlings [48]. the significantly higher transient expression efficiency with Because the flg22 peptide derived from Agrobacterium fla- the AGROBEST than the ABM50 method (Figure 3). gellin is inactive in Arabidopsis [24,34,35] and the flagellin Thus, although C58C1(pTiB6S3ΔT)H remains a strain receptor mutant exhibited similar transformation efficiency causing the least inhibition in seedling root growth as as Col-0 in our seedling assays, the flagellin receptor FLS2 compared to other A. tumefaciens strains, whether the ob- may not be involved in Agrobacterium-triggered plant in- served root growth inhibition results from PTI contribut- nate immune responses and therefore did not compromise ing to reduce transient expression efficiency requires Agrobacterium-mediated transient gene expression. Future future investigation. Other factors in addition to PTI may investigations could examine whether the absence of the contribute to the enhanced transient expression efficiency peptidoglycan receptor [49] or yet-to-be identified recep- by AGROBEST. tors in recognizing additional MAMPs such as polysac- C58C1(pTiB6S3ΔT)H has been known to achieve high charides [50] could increase the transformation efficiency transformation efficiency in several plant species including in seedling roots. Arabidopsis, but the underlying mechanism is not known. The nomenclature of Agrobacterium strains used in plant Key factors for high transient transformation/expression transformation experiments is often simplified, which efficiency causes confusion and could sometimes be misleading. During this course of our method development, we also C58C1(pTiB6S3ΔT)H is often simplified as C58C1 in uncovered new factors critical for the high transient trans- the plant community. C58C1 is in fact named after curing formation/expression efficiency in Arabidopsis seedlings. pTiC58 from the wild-type virulent strain C58, and rifam- One factor is the addition of AB salts in MS medium buff- picin (Rif)-resistant strains are selected from C58C1 for ered with acidic pH 5.5 during Agrobacterium infection, convenient use to acquire various disarmed Ti which allows for significantly higher transient expression transferred from different Agrobacterium strains [52,53]. efficiency than in MS medium alone. Another break- Therefore, C58C1(pTiB6S3ΔT)H is a Rif-resistant C58C1 through is the use of the disarmed A. tumefaciens strain harboring the octopine-type Ti plasmid pTiB6S3 with the C58C1(pTiB6S3ΔT)H, which offers the highest transient removal of the T-DNA region [31] and containing a expression efficiency with the least adverse impact on pCH32 helper plasmid with increased expression of plant growth over other tested strains. Root growth was virulence genes virG and virE2 [32]. GV3101(pMP90) is severely inhibited on infection with other tested A. tume- a C58-derived disarmed strain, in which pMP90 is a faciens strains including the transfer-incompetent ΔvirB2. nopaline-type Ti plasmid, pTiC58, with the removal of These data indicate that the transport of T-DNA and T-DNA [38]. Therefore, in theory, C58C1(pTiB6S3ΔT)H T4SS effectors into plant cells by a virulent C58 strain should share the same chromosomal background with may not suppress host immune responses like that ob- GV3101(pMP90) and only differ in the use of different served in T3SS effectors from Pseudomonas syringae [51]. Ti plasmids and the presence of the helper plasmid We observed that C58C1(pTiB6S3ΔT)H achieved higher pCH32. Future work to determine which genetic factor(s) transient expression efficiency in both Col-0 and efr-1 contribute to increased transient expression efficiency seedlings than other A. tumefaciens strains tested. The with less growth inhibition by C58C1(pTiB6S3ΔT)H will agent also had little impact on root growth inhibition of shed light on understanding the molecular mechanisms infected seedlings by the ABM50 method (Figure 3). The underlying the observed high transient transformation results suggested that the A. tumefaciens strain C58C1 and expression efficiency. (pTiB6S3ΔT)H is the main factor affecting the root growth difference. EFR may play a minor role in root growth in- Conclusions hibition because we observed slightly stronger root growth In this study, we developed a valuable and novel method, inhibition in Col-0 than efr-1 seedlings infected with named AGROBEST, and uncovered the key factors enab- C58C1(pTiB6S3ΔT)H. This finding is consistent with lim- ling this unprecedented high transient transformation and ited root growth inhibition detected in Col-0 seedlings in reporter gene expression efficiency in the immune recep- response to EF-Tu peptide elf18 as compared with strong tor mutant efr-1 and in wild-type Col-0 Arabidopsis seed- root growth inhibition induced by flg22 [47]. The observed lings. The applicability for transient expression of MYB75 inverse association of root growth inhibition and transient in activating downstream gene expression in a Col-0 expression efficiency suggested that C58C1(pTiB6S3ΔT)H background further suggested that AGROBEST may be may circumvent a plant defense barrier to enable high a feasible method to use in examining transcription transient expression levels in Arabidopsis seedlings. factor actions or gain-of-function studies in different However, interestingly, root length was significantly lower Arabidopsis ecotypes/genotypes. Because most plants do in Col-0 and efr-1 seedlings with C58C1(pTiB6S3ΔT)H not harbor EFR, which is only present in Brassicaceae Wu et al. Plant Methods 2014, 10:19 Page 13 of 16 http://www.plantmethods.com/content/10/1/19

[24], the established method may be applicable in other acetosyringone (AS; 0, 50 or 200 μM) without antibiotics, plant species. This fast, sensitive, and quantitative then shaken (220 rpm) at 28°C for 12–16 hr. Before infec- assay was routinely used with culture plates, which are tion, A. tumefaciens cells were pelleted and re-suspended easily scaled up for quick and systematic screens. Im- in desired co-cultivation liquid media to OD600 0.02. The portantly, this method nicely compliments the commonly growth medium of Arabidopsis seedlings was replaced used Arabidopsis mesophyll-protoplast transfection with 1 ml A. tumefaciens cells freshly prepared above and [15,16] and Agrobacterium-mediated leaf infiltration in incubated in the same growth room until further analysis. N. benthamiana [17] for gene functional studies and Three-day-old seedlings were treated with 10 μM DEX for provides advantages for its high reproducibility with- 1 day before infection for the following 3 days. When the out advanced skills. Furthermore, AGROBEST may be removal of Agrobacterium cells was required, co-cultivation an alternative method for evaluating Agrobacterium medium was removed after the chosen infection time and virulence and discovering and dissecting gene func- replaced with 1 ml freshly prepared MS medium containing tions involved in various steps of Agrobacterium- 100 μM Timentin and incubated for additional days before mediated DNA transfer. The method may help unravel analysis. The procedures for the seedling transient trans- the mechanisms underlying Agrobacterium infection in formation assay using the method optimized by Marion unwounded cells/tissues. et al. and FAST Method developed by Li et al. were per- formed [26,27] and described in Additional file 4: Methods Methods S1. Unless indicated, 10 seedlings grown in each well were Materials and growth condition infected and 3 biological repeats were performed in each Strains, plasmids, and primer sequences used in this independent experiment. study are in Additional file 2: Table S2 and Additional file 3: Table S3. The bacterial growth conditions and Plant RNA extraction and quantitative RT-PCR procedures for plasmid and mutant constructions are RNA was extracted from Arabidopsis seedlings as de- described in Additional file 4: Methods S1. Arabidopsis scribed [55]. An amount of 4 μg total RNA was used to thaliana plants included ecotype Columbia-0 (Col-0), synthesize first-strand cDNA with SuperScript III Re- Wassilewskija (Ws-2), T-DNA insertion mutants efr-1 verse Transcriptase (Invitrogen) and oligo dT primer. (SALK_044334) and fls2 (SALK_093905) and the DEX- Quantitative PCR involved the Applied Biosystems inducible AvrPto transgenic line generated in a Col-0 QuantStudio 12 K Flex Real Time PCR machine and background were obtained from the Arabidopsis Bio- Power SYBRR Green PCR Master Mix (Invitrogen). Ara- logical Resource Center (Ohio). Seeds were sterilized in bidopsis ACTIN 2 (At3g18780) or UBC21 (At5g25760) 50% bleach (v/v) containing 0.05% Triton X-100 (v/v) was an internal control. for 10 min, rinsed 5 times with sterile water, and incu- bated at 4°C for 3 days. For germination, 10 seeds were GUS staining and activity assays transferred to 1 ml 1/2 MS liquid medium (1/2 MS salt Seedlings were stained with 5-bromo-4-chloro-3-indolyl supplemented with 0.5% sucrose (w/v), pH 5.5 [pH ad- glucuronide (X-Gluc) at 37°C for 6 hr unless indicated justed to 5.7 by KOH but pH 5.5 after autoclaving], in or quantified with a fluorescence substrate (4-methylum- each well of a 6-well plate. Germination and growth belliferyl-β-D-glucuronide [MUG]) as described [56]. For took place in a growth room at 22°C under a 16-hr/8-hr MUG assay, fluorescence was determined using a 96 light–dark cycle (75 μmol m-2 s-1). microtiter-plate reader (Bio-Tek Synergy Mx, 356 nm excitation 455 nm emission with ±20 nm filter) and cal- Agrobacterium infection in Arabidopsis seedlings culation of specific GUS enzyme activity was based on For AGROBEST infection assay, A. tumefaciens was freshly the standard curve of 0.5–500 pmole (0.5, 5, 50 and 500 streaked out from -80°C glycerol stock onto a 523 agar pmole) 4-MU standards obtained from the same micro- plate for 2-day incubation at 28°C. A fresh single colony titer plate. For relative GUS activity, the fluorescence from the plate was used to inoculate 5 ml of 523 liquid signal value was normalized by an equal amount of pro- medium containing appropriate antibiotics for shaking teins with subtraction of the background fluorescence (220 rpm) at 28°C for 20–24 hr. For pre-induction of A. signal detected by the mock control. tumefaciens vir gene expression, A. tumefaciens cells were pelleted and re-suspended to OD600 0.2 in various liquid Confocal microscopy media including LB, LB-MES (LB with 10 mM MES, Fluorescence signals were observed by use of a Zeiss pH 5.7) [53,54] or AB-MES (17.2 mM K2HPO4,8.3mM LSM 510 Meta Confocal microscope. Venus signals were NaH2PO4,18.7mMNH4Cl, 2 mM KCl, 1.25 mM MgSO4, observed at 488-nm excitation with an HFT 488/514-nm 100 μMCaCl2,10μM FeSO4, 50 mM MES, 2% glucose filter and emission with NTF 515- and BP 505- to 530-nm (w/v), pH 5.5) [37] with different concentrations of filters. RFP signals were observed at 488-nm excitation Wu et al. Plant Methods 2014, 10:19 Page 14 of 16 http://www.plantmethods.com/content/10/1/19

with an HFT 405/488-nm filter and emission with NFT normalized to the protein amount of each sample quanti- 545 and LP 650 filters. fied by the Bradford protein assay (Bio-Rad).

Transient expression of MYB75 and anthocyanin content Additional files assay Four-day-old seedlings were infected with A. tumefaciens Additional file 1: Table S1. Transient transformation efficiency of strain C58C1(pTiB6S3ΔT)H carrying the control or shoots and roots of Arabidopsis Col-0 and efr-1 seedlings. MYB75-expressing binary vector in ABM-MS liquid Additional file 2: Table S2. Bacterial strains and plasmids. medium for 3 days. The co-cultivation medium was then Additional file 3: Table S3. Primer information. replaced with 1 ml fresh MS medium (1/2 MS, 2% su- Additional file 4: Methods S1. Methods for bacterial strains and plasmids and infection method by Li et al. (FAST method) and by Marion et al. crose (w/v), pH adjusted to 5.7 by KOH but pH 5.5 after autoclaving) containing 100 μM Timemtin and then in- Competing interests cubated for 3 days. For anthocyanin content assay, seed- The authors declare that they have no competing interests. lings were blot-dried briefly, weighed, ground into powder with liquid nitrogen and mixed with 1 ml extrac- Authors’ contributions HYW participated the experimental designs, performed most of the tion buffer (0.12 M HCl, 18% isopropanol (v/v)). The mix- experiments, analyzed data, and drafted the manuscript. KHL and WLC ture was boiled for 90 sec and centrifuged at 16000 × g for participated in method optimization. YCW and JFW performed experiments. 15 min. The supernatant was collected and measured at CYC participated in data analysis. SHW and JS participated in experimental designs and helped drafting the manuscript. EML conceived of the study, OD535 (A535) and OD650 (A650). Anthocyanin content was participated in method optimization and experimental designs, coordinated calculated as A535 - (2.2 × A650)/fresh weight (g) [57]. the project, and wrote the manuscript. All authors read and approved the final manuscript. Transient expression of GI::LUC2 and bioluminescence Acknowledgements measurement The authors thank Hau-Hsuan Hwang, Lay-Sun Ma, Jer-Sheng Lin, and Po-Yuan Four-day-old seedlings were infected with A. tumefaciens Shih for discussion and critical reading of the manuscript; and Yajie Niu and strain C58C1(pTiB6S3ΔT)H carrying p1390-GI::LUC2 or Hoosun Chung for preliminary transient expression tests on different seedling ages. We thank Dr. Inhwan Hwang for NLS-RFP; Drs. Stanton Gelvin and empty vector (pCAMBIA1390) in ABM-MS co-cultivation Lan-Ying Lee for pBISN1, Venus-intron and BiFC vectors; and Ms. Mei-Jane medium. At 3 dpi, each seedling was transferred to MS Fang from the Cell Biology Core Laboratory at the Institute of Plant and medium (1/2 MS, pH adjusted to 5.7 by KOH but pH 5.5 Microbial Biology, Academia Sinica, for excellent technical support on confocal microscopy. This work was supported by research grants from the National after autoclaving) containing 100 μMTimentinand Science Council of Taiwan (NSC 99-2918-I-001-005 and NSC 101-2321-B-001-033 0.5 mM luciferin in a black 96-well plate. Bioluminescence to E. M. Lai, NSC100-2311-B-001-028-MY3 to S. H. Wu) and the US National activity was measured and analyzed as described [43]. Institutes of Health (R01GM60493 and R01GM70567 to J. Sheen). Author details Luciferase activity assay 1Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, 2 Arabidopsis seedlings after infection were surface steril- Taiwan. Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan. 3Department of and ized with 1% bleach (0.05% sodium hypochlorite) for 5– Center for Computational and Integrative Biology, Massachusetts General 10 min and washed with sterile water 3 times to remove Hospital, Boston, MA 02114, USA. 4Department of Genetics, Harvard Medical 5 bacteria before assay. The washing step is essential to School, Boston, MA 02114, USA. Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA 23284, USA. minimize the background signals expressed in bacteria be- cause of the use of intron-less LUC2 reporter. For photog- Received: 7 March 2014 Accepted: 28 May 2014 raphy, 10 seedlings infected by each method were placed Published: 18 June 2014 in a clean 15-cm square Petri dish and covered with References 100 μl 1 mM luciferin. Luciferase intensity was imaged by 1. Dandekar AM, Fisk HJ: Plant transformation: Agrobacterium-mediated use of the XENOGEN IVIS lumina system with 5-sec ex- gene transfer. Methods Mol Biol 2005, 286:35–46. 2. Gelvin SB: Agricultural biotechnology: gene exchange by design. Nature posure time. Bioluminescence assay involved the luciferase 2005, 433:583–584. assay system (Promega). Briefly, 10–15 seedlings after a 3. Tzfira T, Citovsky V: Agrobacterium-mediated genetic transformation of washing were blot-dried with tissue paper before being plants: biology and biotechnology. Curr Opin Biotechnol 2006, 17:147–154. 4. Gelvin SB: Agrobacterium-mediated plant transformation: the biology frozen with liquid nitrogen and stored at -80°C. Seedlings behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 2003, 67:16–37. were ground into fine powder by liquid nitrogen, mixed 5. Gelvin SB: Plant proteins involved in Agrobacterium-mediated genetic with 300 μl cell-culture lysis reagent (Promega), and cen- transformation. Annu Rev Phytopathol 2010, 48:45–68. 6. Gelvin SB: Traversing the cell: Agrobacterium T-DNA’s journey to the host trifuged at 16000 × g for 10 min at 4°C. Supernatant was genome. Front Plant Sci 2012, 3:52. 100× diluted with cell-culture lysis reagent. In total, 20 μl 7. McCullen CA, Binns AN: Agrobacterium tumefaciens and plant cell cell lysate was mixed with 100 μl Luciferase Assay Reagent interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol 2006, 22:101–127. and the signal was detected by use of lumat LB 9507 8. Fronzes R, Christie PJ, Waksman G: The structural biology of type IV (Berthold Technologies). The bioluminescence signal was secretion systems. Nat Rev Microbiol 2009, 7:703–714. Wu et al. Plant Methods 2014, 10:19 Page 15 of 16 http://www.plantmethods.com/content/10/1/19

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doi:10.1186/1746-4811-10-19 Cite this article as: Wu et al.: AGROBEST: an efficient Agrobacterium- mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods 2014 10:19.

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